Heteromerization of Gs with Gi protein-coupled receptors has been suggested to be necessary to sustain the canonical Gi-Gs antagonistic interaction at adenylyl cyclase (AC). These heteromers have a tetrameric quaternary structure, composed by two homodimers each one coupled to its corresponding G protein. We describe the heterotetramer formed by the Gi-coupled μ-opioid receptor (MOR) and the Gs-coupled corticotropin releasing factor CRF1 receptor (CRF1R), which also sustains a canonical interaction at AC and reciprocal allosteric interactions between MOR and CRF1R ligands. In addition, we found that CRF1R can also couple to Gq proteins in the MOR-CRF1R heteromer, providing the frame for also canonical Gi-Gq antagonistic interactions that include other effectors, such as phospholipase C and its Ca2 + -dependent signaling, and which control glutamate release in the central amygdala (CeA). The specific pharmacodynamic properties of the MOR-CRF1R heteromer, including its sensitivity to S-methadone, as well as its localization in the CeA suggest it might represent a significant pharmacological target for the analgesic, antistressor and antidepressant effects of opioids and the hyperalgesia of opioid withdrawal.

Several dopaminergic compounds, including the clinically used pramipexole, are labelled as preferential dopamine D3 receptor (D3R) agonists based on their moderately higher affinity for the D3R versus other D2-like receptor subtypes. In rodents, these compounds typically produce locomotor depression with low doses and locomotor activation with higher doses, which has been assumed to be mediated by presynaptic D3Rs and postsynaptic striatal D2Rs, respectively. However, studies with selective pharmacological and genetic blockade of each dopamine receptor subtype suggest opposite roles. We address this apparent conundrum by performing a comprehensive in vitro, in vivo and ex vivo pharmacological comparison of several preferential D3R agonists. Their differential properties reveal that their locomotor activating effects in mice are dependent on the striatal postsynaptic D3Rs forming heteromers with D1Rs, via their ability to potentiate β-arrestin recruitment by the D1R in the D1R-D3R heteromer. The results also indicate that the locomotor depressant effects are largely dependent on their ability to activate presynaptic D2Rs. More broadly, it is demonstrated that locomotor activity in mice depends on β-arrestin recruitment by the D1R in the striatal D1R-D3R heteromer. These results can have implications for the treatment of L-dopa-induced dyskinesia and Restless Legs Syndrome.

Activation of cannabinoid CB1 receptors (CB1R) by agonists induces analgesia but also induces cognitive impairment through the heteromer formed between CB1R and the serotonin 5HT2A receptor (5HT2AR). This side effect poses a serious drawback in the therapeutic use of cannabis for pain alleviation. Peptides designed from the transmembrane helices of CB1R, which are predicted to bind 5HT2AR and alter the stability of the CB1R-5HT2AR heteromer, have been shown to avert CB1R agonist-induced cognitive impairment while preserving analgesia. Using these peptides as templates, we have now designed nonpeptidic small molecules that prevent CB1R-5HT2AR heteromerization in bimolecular fluorescence complementation assays and the heteromerization-dependent allosteric modulations in cell signaling experiments. These results provide proof-of-principle for the design of optimized ligand-based disruptors of the CB1R-5HT2AR heteromer, opening new perspectives for in vivo studies.

Cereblon/CRBN is a substrate-recognition component of the Cullin4A-DDB1-Roc1 E3 ubiquitin ligase complex. Destabilizing mutations in the human CRBN gene cause a form of autosomal recessive non-syndromic intellectual disability (ARNSID) that is modelled by knocking-out the mouse Crbn gene. A reduction in excitatory neurotransmission has been proposed as an underlying mechanism of the disease. However, the precise factors eliciting this impairment remain mostly unknown. Here we report that CRBN molecules selectively located on glutamatergic neurons are necessary for proper memory function. Combining various in vivo approaches, we show that the cannabinoid CB 1 receptor (CB 1 R), a key suppressor of synaptic transmission, is overactivated in CRBN deficiency-linked ARNSID mouse models, and that the memory deficits observed in these animals can be rescued by acute CB 1 R-selective pharmacological antagonism. Molecular studies demonstrated that CRBN interacts physically with CB 1 R and impairs the CB 1 R-G i/o -cAMP-PKA pathway in a ubiquitin ligase-independent manner. Taken together, these findings unveil that CB 1 R overactivation is a driving mechanism of CRBN deficiency-linked ARNSID and anticipate that the antagonism of CB 1 R could constitute a new therapy for this orphan disease.

In “Advanced Enzymology” at the University of Barcelona, we teach various methods for identifying uni-uni irreversible reactions. Kinetic constants such as maximum velocity (Vmax) and Michaelis constant (Km) are calculated using non-linear regression with the Michaelis-Menten equation. However, if a computer is not available, students must calculate these kinetic parameters using linear regressions with Lineweaver-Burk, Eadie-Hofstee, and Hanes-Woolf plots. Michaelis-Menten equation: v = (Vmax [S]) / (Km + [S]), Lineweaver-Burk equation: 1/v = (Km/Vmax) (1/[S]) + 1/Vmax, Eadie-Hofstee equation: v = – Km (v/[S]) + Vmax, Hanes-Woolf equation: [S]/v = (1/Vmax) [S] + (Km/Vmax). The objective of this work was that students explore various methods for calculating kinetic constants and determine the most effective approach. A problem was prepared with data on substrate concentrations ([S]) and reaction rates (v). Data points with lower substrate concentration, and also with lower velocities had higher errors. The Michaelis-Menten plot provided only an approximate estimation of the kinetic constants, as it is not a linear graph. The Lineweaver-Burk plot generated maximum velocities that were too low due to the higher errors for low substrate concentrations, rendering impossible values of the kinetic parameters. The Eadie-Hofstee plot provided better results, as the values of v/[S] compensated their errors. Furthermore, kinetic constants were obtained directly from the slope and intersection of the line equation. The Hanes-Woolf plot also produced correct values for kinetic parameters, since high substrate concentrations were less erroneous than lower ones. After completing this computer class, students were very satisfied and learned the different methods for obtaining kinetic parameters. Non-linear regression is undoubtedly the most accurate method for obtaining kinetic parameters values. However, in the absence of computers, the Eadie-Hofstee plot is the best and most commonly used plot in kinetic papers.

(R,S)-methadone ((R,S)-MTD) is a µ-opioid receptor (MOR) agonist comprised of (R)-MTD and (S)-MTD enantiomers. (S)-MTD is being developed as an antidepressant and is considered an N-methyl-D-aspartate receptor (NMDAR) antagonist. We compared the pharmacology of (R)-MTD and (S)-MTD and found they bind to MORs, but not NMDARs, and induce full analgesia. Unlike (R)-MTD, (S)-MTD was a weak reinforcer that failed to affect extracellular dopamine or induce locomotor stimulation. Furthermore, (S)-MTD antagonized motor and dopamine releasing effects of (R)-MTD. (S)-MTD acted as a partial agonist at MOR, with complete loss of efficacy at the MOR-galanin Gal 1 receptor (Gal 1 R) heteromer, a key mediator of the dopaminergic effects of opioids. In sum, we report novel and unique pharmacodynamic properties of (S)-MTD that are relevant to its potential mechanism of action and therapeutic use. One-sentence summary: (S)-MTD, like (R)-MTD, binds to and activates MORs in vitro, but (S)-MTD antagonizes the MOR-Gal 1 R heteromer, decreasing its abuse liability.

The functional role of the dopamine D 4 receptor (D 4 R) and its main polymorphic variants has become more evident with the demonstration of heteromers of D 4 R that control the function of frontal cortico-striatal neurons. Those include heteromers with the α 2A adrenoceptor (α 2A R) and with the D 2 R, localized in their cortical somato-dendritic region and striatal nerve terminals, respectively. By using biophysical and cell-signaling methods and heteromer-disrupting peptides in mammalian transfected cells and rat brain slice preparations, here we provide evidence for a new functionally relevant D 4 R heteromer, the α 1A R-D 4 R heteromer, which is also preferentially localized in cortico-striatal glutamatergic terminals. Significant differences in allosteric modulations between heteromers of α 1A R with the D 4.4 R and D 4.7 R polymorphic variants could be evidenced with the analysis of G protein-dependent and independent signaling. Similar negative allosteric modulations between α 1A R and D 4 R ligands could be demonstrated for both α 1A R-D 4.4 R and α 1A R-D 4.7 R heteromers on G protein-independent signaling, but only for α 1A R-D 4.4 R on G protein-dependent signaling. From these functional differences, it is proposed that the D 4.4 R variant provides a gain of function of the α 1A R-mediated noradrenergic stimulatory control of cortico-striatal glutamatergic neurotransmission, which could result in a decrease in the vulnerability for impulse control-related neuropsychiatric disorders and increase in the vulnerability for posttraumatic stress disorder.

Cannabinoids exert pleiotropic effects on the brain by engaging the cannabinoid CB1 receptor (CB1R), a presynaptic metabotropic receptor that regulates key neuronal functions in a highly context-dependent manner. We have previously shown that CB1R interacts with growth-associated protein of 43 kDa (GAP43) and that this interaction inhibits CB1R function on hippocampal excitatory synaptic transmission, thereby impairing the therapeutic effect of cannabinoids on epileptic seizures in vivo. However, the underlying molecular features of this interaction remain unexplored. Here, we conducted mechanistic experiments on HEK293T cells co-expressing CB1R and GAP43 and show that GAP43 modulates CB1R signalling in a strikingly selective manner. Specifically, GAP43 did not affect the archetypical agonist-evoked (i) CB1R/Gi/o protein-coupled signalling pathways, such as cAMP/PKA and ERK, or (ii) CB1R internalization and intracellular trafficking. In contrast, GAP43 blocked an alternative agonist-evoked CB1R-mediated activation of the cytoskeleton-associated ROCK signalling pathway, which relied on the GAP43-mediated impairment of CB1R/Gq/11 protein coupling. GAP43 also abrogated CB1R-mediated ROCK activation in mouse hippocampal neurons, and this process led in turn to a blockade of cannabinoid-evoked neurite collapse. An NMR-based characterization of the CB1R-GAP43 interaction supported that GAP43 binds directly and specifically through multiple amino acid stretches to the C-terminal domain of the receptor. Taken together, our findings unveil a CB1R-Gq/11-ROCK signalling axis that is selectively impaired by GAP43 and may ultimately control neurite outgrowth.

The Montessori method was an educational model devised at the beginning of the 20th century by the Italian pedagogue, physician, psychologist, and psychiatrist María Montessori (1870-1952). She observed that her students activated their intelligence and developed their personality by carrying out manual activities. In recent years, one of the activities that she used, gamification, is currently booming in teaching. Games based in words or letters are often used in language learning, either in presential lessons or in mobile applications. In our teaching innovation group (GINDOC-UB/180), we proposed to adapt those word games using codes so that students could achieve a self-learning on the nomenclature of biomolecules, thus improving their knowledge in metabolism. These code-based games were proposed to Biochemistry students at the Chemistry degree of the Universitat de Barcelona. Games were posted on the Virtual Campus using Moodle. A code is part of a communicative system, and it is defined as a set of elements that allows a message to be decoded. In biochemistry, the best-known code is the genetic code that translates 3 nucleotides to an amino acid. The code allows the students to see that it is a degenerated code, and it was the first code-based game used. Nevertheless, only 20 amino acids are involved, and some letters are missing. Thus, we also used other codes such as numeric codes, Morse code, Braille system code, or codes from other alphabets (Japanese katakana or hiragana, Cyrillic alphabet, Arabic alphabet, or runic alphabet). In this work some examples of the games that can be proposed to the students are shown. Proposed games were highly valued by students and allowed a self-learning on the proposed biomolecules. Games based on the genetic code were easier and more enjoyable for the students to solve, and they provided them with additional biochemical knowledge of protein translation.

Introducción y problema: Los juegos son muy apreciados por la población en general, así que realizamos una búsqueda de diversos juegos con la finalidad de utilizarlos para reforzar los conocimientos en bioquímica de los alumnos del grado de Química de la Universidad de Barcelona. Los juegos de los libros de pasatiempos están basados en dos posibilidades: números y letras. Entre los juegos de letras los más frecuentes son los crucigramas, autodefinidos, palabras cruzadas y sopas de letras. Metodología: Para reforzar los conocimientos de los estudiantes en Bioquímica, realizamos una búsqueda de juegos de palabras usados en el aprendizaje de gramática de idiomas, para luego adaptarlos a la nomenclatura y estructura de biomoléculas en Bioquímica. Siguiendo una aproximación conductista, utilizamos el modelo de Dick y Carey. Resultados y discusión: Los juegos más sencillos sobre palabras se basan en buscar dentro de un conjunto de palabras, aquellas que tengan alguna parte en común. En este sentido, implementamos juegos de rimas, en los que las palabras terminan en –ina (terminación frecuente de los aminoácidos, bases nitrogenadas, nucleósidos y algunas proteínas), en –osa (terminación frecuente de los hidratos de carbono), o en –ico / –ato (terminación frecuente de los ácidos grasos y otros ácidos intermediarios del metabolismo, o de sus sales correspondientes). Así pues, los alumnos pueden observar estas características en la nomenclatura. Por otro lado, también implementamos juegos de palabras incompletas que se basan en completar los nombres de los metabolitos utilizando grupos de letras a escoger. Los juegos se clasificaron en 4 grupos y se presentan algunos ejemplos de estos. Respecto a la dificultad, los juegos tipo dominó son los más difíciles de resolver, pues requieren conocer tanto la nomenclatura como la estructura de las biomoléculas. Conclusiones: Nuestra propuesta incluye diversos juegos de palabras que permiten reforzar conocimientos de Bioquímica.

APRESENTAÇÃOLa colección "Estudios Teórica-Metodológicas

Knight tour is a mathematical problem that was first solved by Leonhard Euler (1707-1785). The problem consists in finding if it is possible that the knight piece of chess can tour through all the boxes of a chess grid, passing only once through each box. Bishop piece can only move to diagonal boxes of one color, and pawns can only move in one column. It can be easily seen that king, queen and rook can move through all the boxes. But it was not clear that the knight could move all through. Euler found several solutions for the 8x8 grid, and he numbered all the boxes of the grid in the order through which the knight passed. Surprisingly, he obtained some solutions with semi-magic squares, in which the sum of all the numbers of each row and column was the same. Nevertheless, diagonals didn’t sum the same, as happens in the magic squares. One of the typical games in word game’s books is the knight tour, with a 5x5 grid, that hides a 25-syllable sentence. The objective of this work was to study other grids, to obtain different sentences length with 3x3 (9-syllables), 4x4 (16-syllables), 5x5 (25 syllables), 6x6 (36 syllables) or 7x7 (49 syllables) grids. Thus, this game would be more versatile and could be used more extensively than limiting it to 25-syllable sentences.

The type-1 cannabinoid receptor (CB1R) is widely expressed in excitatory and inhibitory nerve terminals, and by suppressing neurotransmitter release, its activation modulates neural circuits and brain function. While the interaction of CB1R with various intracellular proteins is thought to alter receptor signaling, the identity and role of these proteins are poorly understood. Using a high-throughput proteomic analysis complemented with an array of in vitro and in vivo approaches in the mouse brain, we report that the C-terminal, intracellular domain of CB1R interacts specifically with growth-associated protein of 43kDa (GAP43). The CB1R-GAP43 interaction occurs selectively at mossy cell axon boutons, which establish excitatory synapses with dentate granule cells in the hippocampus. This interaction impairs CB1R-mediated suppression of mossy cell to granule cell transmission, thereby inhibiting cannabinoid-mediated anti-convulsant activity in mice. Thus, GAP43 acts as a synapse type-specific regulatory partner of CB1R that hampers CB1R-mediated effects on hippocampal circuit function.

A G protein-coupled receptor heteromer that fulfills the established criteria for its existence in vivo is the complex between adenosine A2A (A2AR) and dopamine D2 (D2R) receptors. Here, we have designed and synthesized heterobivalent ligands for the A2AR-D2R heteromer with various spacer lengths. The indispensable simultaneous binding of these ligands to the two different orthosteric sites of the heteromer has been evaluated by radioligand competition-binding assays in the absence and presence of specific peptides that disrupt the formation of the heteromer, label-free dynamic mass redistribution assays in living cells, and molecular dynamic simulations. This combination of techniques has permitted us to identify compound 26 [KDB1 (A2AR) = 2.1 nM, KDB1 (D2R) = 0.13 nM], with a spacer length of 43-atoms, as a true bivalent ligand that simultaneously binds to the two different orthosteric sites. Moreover, bioluminescence resonance energy transfer experiments indicate that 26 favors the stabilization of the A2AR-D2R heteromer.

A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D1 and D3 receptors (D1R and D3R) influences the pharmacological properties of three structurally similar selective dopamine D3R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4–116. By using D1R-D3R heteromer-disrupting peptides, it could be demonstrated that the three D3R ligands display different D1R-D3R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D1R-mediated signaling in the D1R-D3R heteromer; PG01037, acting as a D3R antagonist cross-antagonized D1R-mediated signaling in the D1R-D3R heteromer; and VK4–116 specifically acted as a ß-arrestin-biased agonist in the D1R-D3R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D3R ligands that are dependent on D1R-D3R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D3R ligands in vivo. The results supported the involvement of D1R-D3R heteromers in the locomotor activation by D1R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D1R-D3R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson’s disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development.

The functional and pharmacological significance of the dopamine D4 receptor (D4R) has remained the least well understood of all the dopamine receptor subtypes. Even more enigmatic has been the role of the very prevalent human DRD4 gene polymorphisms in the region that encodes the third intracellular loop of the receptor. The most common polymorphisms encode a D4R with 4 or 7 repeats of a proline-rich sequence of 16 amino acids (D4.4R and D4.7R). DRD4 polymorphisms have been associated with individual differences linked to impulse control-related neuropsychiatric disorders, with the most consistent associations established between the gene encoding D4.7R and attention-deficit hyperactivity disorder (ADHD) and substance use disorders. The function of D4R and its polymorphic variants is being revealed by addressing the role of receptor heteromerization and the relatively avidity of norepinephrine for D4R. We review the evidence conveying a significant and differential role of D4.4R and D4.7R in the dopaminergic and noradrenergic modulation of the frontal cortico-striatal pyramidal neuron, with implications for the moderation of constructs of impulsivity as personality traits. This differential role depends on their ability to confer different properties to adrenergic α2A receptor (α2AR)-D4R heteromers and dopamine D2 receptor (D2R)-D4R heteromers, preferentially localized in the perisomatic region of the frontal cortical pyramidal neuron and its striatal terminals, respectively. We also review the evidence to support the D4R as a therapeutic target for ADHD and other impulse-control disorders, as well as for restless legs syndrome.